Milk fat globule membrane material:

challenges and opportunities for the

food industry
Prof. Dr. ir. Koen Dewettinck
UGent, Belgium
CEO UGent Cacaolab bvba
1

Milk microstructure (x 50,000)

Membrane nanostructure model

(Dewettinck et al., 2008. International Dairy Journal )

Occurrence of MFGM-material
During processing of milk:

Disruption of MFGM
MFGM fragments exists as such in processed dairy products

(Wooding et al., 1975. Journal of Dairy Research)

Occurrence of MFGM-material
Centrifuged buttermilk (8500)

(Wooding et al., 1975. Journal of Dairy Research)

Outline

Purification/analysis of MFGM
Nutritional properties of MFGM
Technological properties of MFGM
Future trends and challenges

Outline

Purification/analysis of MFGM
Nutritional properties of MFGM
Technological properties of MFGM
Future trends and challenges

 Purification of MFGM fragments

From Raw Milk
From Dairy Industrial by-products

From raw milk by washing method

Milk
Separating
fat globules
buffers ?

1st washing

volumes ?

2nd washing

temperatures ?

3rd washing

Discharged
Washing buffer

Washed
cream
Churning
Buttermilk

Butter
Melting +
Centrifuging
Butter oil

Butter
serum

MFGM
suspension

Lyophilizing
MFGM
material

Store at <-20oC
9

From raw milk by washing method

Milk
Deionized water
At 9/1 volume
Temp: lower or higher?

Separating
fat globules
1st washing
2nd

washing

3rd

washing
Washed
cream

1st discharged water

2nd discharged water
3rd discharged water
MFGM proteins

Caseins
Whey proteins
(Le et al., 2009. Journal of Dairy Science )

10

 Purification of MFGM fragments

From Raw Milk
From Dairy Industrial by-products

11

Isolation of MFGM from

dairy by-products

Butter serum
(BS)
Sodium
citrate

Buttermilk
(BM)

Dissociating
casein micelles

Coagulating
Caseins

Casein coagulate
fresh cheese

By citric acid

Deionized
water
0.22m

45 C
pH 7.6

Permeate

BM-MFGM

(Rombaut et al., 2006c. Journal of Dairy Science )

MF + DF

Deionized
water

MF + DF

Buttermilk whey
(BMW)

0.22m

45 C
pH 7.6

Permeate

BMW-MFGM

(Rombaut et al., 2007a. Journal of Dairy Science )

12

Crossflow microfiltration
Lactose
Minerals
Whey
MFGMproteins

13

(Dia)Filtration of acid whey

Whey

1.63% PL/DM

Filtrate 1

Retentate 1

5.86% PL/DM

Filtrate 3

Retentate 3

10.36% PL/DM

(Rombaut et al., 2007a. Journal of Dairy Science )

14

Thermocalcic aggregation of whey

Whey/
buttermilk whey
Thermocalcic
aggregation

Whey

Supernatant

1.63% PL/DM

Supernatant 2 Coagulate

11.18% PL/DM

Centrifugation/
filtration
Pellet washing
MFGM isolate

(Rombaut et al., 2007b. Journal of Dairy Science ) 15

Analysis of MFGM polar lipids

Extraction
Liquid-liquid extraction with use of chloroform and methanol is still
widely applied

Analysis
Total phospholipid content: via phosphorous content (several ways)
Analysis of polar lipid species:
Thin-layer chromatography
HPLC-ELSD
31P nuclear magnetic resonance (NMR) spectroscopy
Mass spectrometry: e.g. MALDI-TOF MS, ESI-MS/MS
(Rombaut et al., 2006a. International Dairy Journal )

16

Analysis of MFGM polar lipids

using HPLC-ELSD

Column: Prevail silica 3u 150 x 3.0 mm (Grace Davison)

Total flow rate: 0.5 mL/min, Column oven set at 40 C, sample chamber set at 20 C

Method of Rombaut et al. (2005)

Extracted solvents Chloroform/methanol

Method of Le et al. (2011a)

Chloroform/methanol with adding
CaCl2

Mobile phase

Chloroform/methanol/triethylamine Dichloromethane/methanol/
buffer (pH 3, formic acid)
triethylamine buffer (pH 4.5,
acetic acid)

Total PLs of
MFGM material
(mg/g)

8.95 0.57

9.95 0.23

17

MFGM Polar Lipids profile

Neutral lipids

PE

Phospholipids (~70%)

PE - Phosphatidylethanolamine
PI - Phosphatidylinositol
PS - Phosphatidylserine
PC - Phosphatidylcholine

PI

PS

HPLC-ELSD
18

MFGM Polar Lipids profile

Neutral lipids

PE

Phospholipids (~70%)

PE - Phosphatidylethanolamine
PI - Phosphatidylinositol
PS - Phosphatidylserine
PC - Phosphatidylcholine

PI
Glucer

Laccer
SM
PS

Sphingolipids (~30%)

HPLC-ELSD
Glucosylceramide (Glucer)
Lactosylceramide (Laccer)
Sphingomyelin (SM)

(Le et al., 2011a. Journal of Agricultural and Food Chemistry)

19

Analysis of MFGM proteins

Total (crude) protein: via nitrogen content by Kjeldahl method
Electrophoresis:
1DE: e.g. SDS-PAGE

Separation on molecular weight

MFGM purified

Butter serum

2DE: IEF + SDS-PAGE (from butter serum)

- Separation on charge (iso-electric point)

- Separation in the 2nd dimension on molecular weight

(Rombaut, 2006. PhD dissertation)

20

Proteomic analysis of MFGM proteins

MFGM
material
Defat

Lipids

MFGM
proteins
[1]

[2]

1DE
SDS-PAGE

2DE
IEF/SDS-PAGE

[3]

Tryptic
digestion

In-gel tryptic
digestion of gel
slices or spots

HPLC
fractionation
[3']
A

MALDI-TOF

MALDI-TOF/
TOF

LC-MS/MS

Database search

21

LC-ESI-MS/MS analysis protocol

Separation by
Samples/
Protein mixtures SDS-PAGE

LC-ESI-MS/MS
trypsin

Excision

22

MFGM-associated proteins

A total of 225 proteins identified with 2 peptide matches

116 of the identified proteins have been reported before
25 newly identified MFGM-associated proteins with 5
peptide matches

23

Distribution of MFGM proteins

during dairy processing
Cream
47 proteins

Skim milk
34 proteins

Pooled, 2x washed
cream buttermilk
142 proteins

Pooled, 2x washed
cream butter serum
160 proteins

18
9

2
19
4

32

110

50

18

184
225 MFGM-associated
proteins

(Le et al., 2013. International Dairy Journal)

24

Outline

Purification/analysis of MFGM
Nutritional properties of MFGM
Technological properties of MFGM
Future trends and challenges

(Dewettinck et al., 2008. International Dairy Journal )

25

Nutritional properties of
sphingolipids
Highly bioactive molecules
Hard to digest colon

Ceramidase
Sphingomyelinase
26

Nutritional properties of
sphingolipids
Prevention of colon cancer
Chemopreventive and chemotherapeutic
Tests on rodents (0.025% - 0.5% of the diet)
Intestinal absorption of cholesterol
Mice: reduction of 85% when fed 5% milk SM
Adherence of pathogens on intestinal mucosa

27

Nutritional properties of
phospholipids
Phosphatidylcholine
Supports liver recovery
Good source of choline
(= essential nutrient: important to memory and brain development)
Phosphatidylserine
Attenuation of neuronal effects of ageing (cognitive functions)
Positive effects on
Alzheimer patients (200 mg/day)
Muscular soreness and well-being
Low contribution of dairy products
28

Nutritional properties of
MFGM proteins
Anticancer effects
Breast cancer cell lines
DNA repair
Inhibitor of -glucuronidase (colon cancer)
Positive influence of BTN on autoimmune encephalomyelitis
(multiple sclerosis) and autism
Direct antibacterial effects
XDH/XO
PP3 lactophoricin
Anti-adhesive effects
Glycolipids (MUC 1, PAS 6/7) - H. pylori
29

Nutritional tests with InPulseTM 35

Test on piglets (0.5% InPulse 35/day)

Ratio villi/crypts in duodenum and ileum

30

Nutritional tests with InPulseTM 200

Human test on bowel permeability
in collaboration with Maastricht University
double-blind, randomized, placebo controlled, crossover test setup
10 healthy volunteers, 2 x 5 days
10 g/day
lactulose / rhamnose test with indomethacin
Results
treated persons
no increase of bowel permeability due to indomethacin stress
InPulse 200 promotes bowel integrity under stress conditions
31

Nutritional tests with InPulseTM 200

Test setup with children
in collaboration with Koningin Paola Hospital
test on 253 children 2,5 < x < 6 year
double-blind, placebo controlled
0,5 g/day - 120 days
ASEBA list of questions about behavior
Results
significant difference (p<0,03) in # days of fever (after 6 weeks)
significant improvement of behavior:
internal (p<0.003)
external (p<0.004)
total problem scores (p<0.002)
(Veereman-Wauters et al., 2012. Nutrition)

32

Enzymatic digestion of
MFGM proteins

Protein

Enz. gastric digestion

Pepsin
peptides
pH 2

Enz. intestinal digestion

(1) Tryp + cTryp
Smaller peptides
Peptides (2) Pan + Bile salt
Amino acids
pH 6.5

33

Gastrointestinal digestion of
whole MFGM
1: Whole MFGM
2: 2h with Pepsin
3: + 2.5h Pepsin + Tryp/cTryp

1 2 3

2: 2h with Pepsin
3: + 2.5h Pepsin + Pan/bile salt

2 3

1 2 3

2 3

MUC 1

MUC 1
PAS 3
CD 36
PAS6/7

CB staining

Whole MFGM

PAS staining

CB staining

Whole MFGM

PAS staining

34

Enzymatic digestion of
MFGM proteins
DF MFGM

Whole MFGM
Pepsin

Pepsin

MUC1
PAS3
CD36
PAS6/7

MUC1
PAS3
CD36
Tryp/cTryp
MUC1

Tryp/cTryp

Pancreatic juice/bile salt

MUC1

MUC1
(PAS3)
CD36
PAS 6/7

Resistance of MFGM proteins to proteolytic digestion

Glycosylation of proteins
Hindering effect of lipids

(Le et al., 2012. Journal of Dairy Science)

35

Outline

Purification/analysis of MFGM
Nutritional properties of MFGM
Technological properties of MFGM
Future trends and challenges

36

Technological properties of MGFM

MFGM fragments
efficient emulsifiers
~ original function stabilizing fat globules
MFGM material content influence on emulsion properties

Temperature > 65C emulsifying capacity

denaturation of membrane proteins
association of whey proteins with MFGM

37

Possible applications of MFGM

Bread improver (antistaling agent)

Whipped cream (improving whipping properties of RC)
Chocolate rheology (viscosity lowering)
Instant powders
Antispattering agent in margarine
Liposomes
Source of ceramides for cosmetics

38

Natural MFGM = natural emulsifier

MFGM isolated from raw milk: good

emulsifier
MFGM isolated from industrial sources: not
always superior compared to the material
from which MFGM is isolated (depends on
historical heat treatment)

39

Emulsifying properties of MFGM

16
14

Diluted in SDS

Diluted in water
16

MFGM from BMP

14

10
8
6

12
Sodium caseinate

BMP

Volume (%)

Volume (%)

12

SMP

10
8
6

0
0.01

0
0.01
1000

0.1
1
10
100
Particle diameter (m)

0.1

10

100

1000

Particle diameter (m)

40

Emulsifying properties of MFGM

BMP

MFGM from BMP

16
14
12
10
8
6
SC

50m

50m

SMP

4
2
0
0.01

0.1

50m

10

100

1000

50m

(Phan et al., 2013. International Dairy Journal)

41

Emulsifying capacity
InPulseTM 35 & 200
Test set-up:

1 g powder dissolved in 100 gram Tris-buffer

soy oil is added continuously
homogenization (45C, 200 bar)
emulsifying capacity = % of oil at which the emulsion breaks
down

42

Emulsifying capacity
InPulseTM 35 & 200

400

366

350

% oil added

300

271

270

250
208
200
150
100
50
0
Skim milk powder Buttermilk powder

InPulse 35

InPulse 200

43

Emulsion stability with InPulseTM 200

Test set-up:

1g or 3g powder
10g soy oil
add Tris-buffer to 100g
homogenization (45C, 200 bar)
particle size is measured after 9 days

44

Emulsion stability with InPulseTM 200

10
9
skim milk powder 1%

Volume %

skim milk powder 3%

buttermilk powder 1%

buttermilk powder 3%
InPulse 200 1%

InPulse 200 3%

4
3
2
1
0
0

10

20

30

40

50

60

70

Diameter (D32) (m)

45

Emulsion stability with InPulseTM 200

Test set-up:

Tris buffer with 10% soy oil and 1% powder

homogenization (45C, 200 bar)
after 24 h: centrifugation (5 min at 1000g)
stabilizing capacity = height of cream layer compared to total
height

46

Emulsion stability with InPulseTM 200

30

27

Cream layer (vol%)

25

21
20
15
10
5

0
0

Skim milk powder

Buttermilk powder

InPulse 200

47

MFGM as ingredient in yogurt

75.00

SK12

BM

MFGM

55.00
1.6

45.00

SK12

1.4

BM

MFGM

1.2

0.8
0.6
0.4
0.2

Changed firmness

BM4
MFGM4

BM3
MFGM3

BM2
MFGM2

0
BM1
MFGM1

BM4
MFGM4

SK12

Improved WHC

BM3
MFGM3

BM2
MFGM2

BM1
MFGM1

25.00

Firmness (N)

35.00

SK12

WHC %

65.00

48

MFGM as ingredient in yogurt

1.6

75.00

SK12

BM

MFGM

BM

MFGM

1.2

0.2

Improved WHC

BM4
MFGM4

BM3

BM3
MFGM3

BM3
MFGM3

BM2
MFGM2

BM1
MFGM1

SK12

25.00

0.4

BM4
MFGM4

BM1

SK12

0.6

BM2
MFGM2

35.00

0.8

BM1
MFGM1

45.00

SK12

55.00

Firmness (N)

65.00

WHC %

SK12

1.4

Changed firmness
MFGM1

MFGM3

Modified microstructure(Le et al., 2011b. International Dairy Journal)

49

Outline

Purification/analysis of MFGM
Nutritional properties of MFGM
Technological properties of MFGM
Future trends and challenges

50

Trends and to dos

Detailed quantification of MFGM components in various
dairy products
Confirmation and determination of new health beneficial
properties of MFGM and its components
Nutritional aspects of MFGM in infant foods or pharmacy
Application of MFGM in foodstuffs (margarine, mayonnaise)
combining a technological with a nutritional functionality

51

Main challenges
Stability of MFGM materials and MFGM-enriched products:
- Physicochemical (e.g. optimized processing towards
optimized functionality)
- Chemical: e.g. oxidation stability
Sensorial quality

52

Outline

Publications

1. Le, T. T., G. Debyser, W. Gilbert, K. Struijs, J. Van Camp, T. Van de Wiele, B. Devreese, and K. Dewettinck. 2013.
Distribution and isolation of milk fat globule membrane proteins during dairy processing as revealed by
proteomic analysis. International Dairy Journal 32(2):110-120.
2. Phan, T. T. Q., M. Asaduzzaman, T. T. Le, E. Fredrick, P. Van der Meeren, and K. Dewettinck. 2013. Composition
and emulsifying properties of a milk fat globule membrane enriched material. International Dairy Journal
29(2):99-106.
3. Struijs, K., T. Van de Wiele, T. T. Le, G. Debyser, K. Dewettinck, B. Devreese, and J. Van Camp. 2013. Milk fat
globule membrane glycoproteins prevent adhesion of the colonic microbiota and result in increased bacterial
butyrate production. International Dairy Journal 32(2):99-109.
4. Veereman-Wauters, G., S. Staelens, R. Rombaut, K. Dewettinck, D. Deboutte, R. J. Brummer, M. Boone, and P.
Le Ruyet. 2012. Milk fat globule membrane (INPULSE) enriched formula milk decreases febrile episodes and
may improve behavioral regulation in young children. Nutrition 28(7-8):749-752.
5. Le, T. T., T. Van de Wiele, T. N. H. Do, G. Debyser, K. Struijs, B. Devreese, K. Dewettinck, and J. Van Camp. 2012.
Stability of milk fat globule membrane proteins toward human enzymatic gastrointestinal digestion. Journal
of Dairy Science 95(5):2307-2318.
6. Le, T. T., J. Van Camp, P. A. L. Pascual, G. Meesen, N. Thienpont, K. Messens, and K. Dewettinck. 2011b. Physical
properties and microstructure of yoghurt enriched with milk fat globule membrane material. International
Dairy Journal 21(10):798-805.
7. Le, T. T., J. Miocinovic, T. M. Nguyen, R. Rombaut, J. Van Camp, and K. Dewettinck. 2011a. Improved solvent
extraction procedure and HPLC-ELSD method for analysis of polar lipids from dairy materials. Journal of
Agricultural and Food Chemistry, 59:10407-10413.

Outline

Publications

8. Le, T. T., J. Van Camp, R. Rombaut, F. Van Leeckwyck, and K. Dewettinck. 2009. Effect of washing conditions on
the recovery of milk fat globule membrane proteins during the isolation of milk fat globule membrane from
milk. Journal of Dairy Science 92:3592-3603.
9. Dewettinck, K., R. Rombaut, N. Thienpont, T. T. Le, K. Messens, and J. V. Camp. 2008. Nutritional and
technological aspects of milk fat globule membrane material. International Dairy Journal 18:436-457.
10. Rombaut, R., K. Dewettinck, and J. V. Camp. 2007c. Phospho- and sphingolipid content of selected dairy
products as determined by HPLC coupled to an evaporative light scattering detector (HPLCELSD). Journal of
Food Composition and Analysis 20:308-312.
11. Rombaut, R. and K. Dewettinck. 2007b. Thermocalcic Aggregation of Milk Fat Globule Membrane Fragments
from Acid Buttermilk Cheese Whey. J. Dairy Science 90:2665-2674.
12. Rombaut, R., V. Dejonckheere, and K. Dewettinck. 2007a. Filtration of Milk Fat Globule Membrane Fragments
from Acid Buttermilk Cheese Whey. J. Dairy Science 90:1662-1673.
13. Rombaut, R., V. Dejonckheere, and K. Dewettinck. 2006c. Microfiltration of butter serum upon casein micelle
destabilization. Journal of Dairy Science 89:1915-1925
14. Rombaut, R., J. V. Van Camp, and K. Dewettinck. 2006b. Phospho- and sphingolipid distribution during
processing of milk, butter and whey. International Journal of Food Science and Technology 41(4):435-443.
15. Rombaut, R. and K. Dewettinck. 2006a. Properties, analysis and purification of milk polar lipids. International
Dairy Journal 16:1362-1373.
16. Rombaut, R., J. Van Camp, and K. Dewettinck. 2005. Analysis of phospho- and sphingolipids in dairy products
by a new HPLC method. Journal of Dairy Science 88:482-488.

Acknowledgements

Bllinger Butterei CEO Marc Boone

Royal FrieslandCampina
Dr. Gigi Veereman-Wauters
Prof. John Van Camp & Dr. Karin Struijs
Prof. Bart Devreese
Prof. Tom Vandewiele
PhD students
Dr. Roeland Rombaut
Dr. Thien Trung Le
Msc. Thi Thanh Que Phan
55